Ligand-coupled initiator polymers and methods of use

ABSTRACT

Initiator polymers having an initiator group and a ligand group are provided. The initiator polymers are capable of specifically binding to a receptor on a surface. Using a macromer system, the initiator polymers are useful for the formation of a polymeric matrix on the surface of a material. In particular, initiator polymers are provided that have specificity to pancreatic β cells and can be used to encapsulate cells for transplantation and the treatment of diabetes.

CROSS-REFERENCE TO RELATED APPLICATION

The present non-provisional patent application is a divisional of U.S.patent application Ser. No. 10/412,063, filed on Apr. 10, 2003, now U.S.Pat. No. 7,820,158, entitled LIGAND-COUPLED INITIATOR POLYMERS ANDMETHODS OF USE, which is fully incorporated herein by reference.

FIELD OF THE INVENTION

The current invention relates to compounds useful for forming apolymeric matrix on the surface of a substrate. More specifically, theinvention relates to initiator polymers that can specifically bind to atarget surface and promote formation of a polymeric matrix on thesurface.

BACKGROUND

The use of polymeric material for the encapsulation of cells and tissueoffers great potential for the treatment of diseases and other medicalindications. Particularly useful applications involve utilizingpolymeric material for encapsulating tissues or cells fortransplantation into a patient in order to provide therapy. Althoughvarious techniques for encapsulating mammalian cells have been known fora number of decades and have been used in research settings, only morerecently cell encapsulation technologies have been applied for thepotential treatment of diseases.

Cell encapsulation methods are generally aimed at surrounding a cell orgroup of cells with a material barrier in order to protect thetransplanted encapsulated cells from host immune rejection. The materialbarrier around the cells ideally allows the cells to remain viable andto function properly in order to provide therapeutic value to the host.In order to perform this function, the material that is used toencapsulate the cells, which typically includes a polymeric compound,should be resistant to biodegradation and should be sufficientlypermeable to allow for diffusion of cellular waste products, nutrients,and molecules involved in cellular responses. Preferably, the materialbarrier is not permeable to certain host molecules, such asimmunoglobulins and complement factors that could contribute to thedestruction of the foreign cells.

Advances in cell encapsulation technologies have been focused onimproving the permselectivity, mechanical properties, immuneprotectivity, and biocompatibility of the material barrier that isformed around the cells. Various micro- and macroencapsulationtechniques, including microencapsulation by polyelectrolytecomplexation, thermoreversible gelation, interfacial precipitation,interfacial polymerization, and flat sheet and hollow fiber-basedmacroencapsulation have been studied and are reviewed by Uludag et al.Adv. Drug Deliv. Rev. 42:29-64 (2000).

One commonly used method for the encapsulation of cells is the alginatecrosslinking method, which utilizes polyanionic alginate andpolycationic polylysine polymers. Encapsulation by the alginate methodtypically occurs by the crosslinking of alginate via the Ca²⁺ ion andthe interaction of polylysine with the alginate molecules.Unfortunately, there are a number of problems associated with thisapproach to cell encapsulation. Such problems include the swelling ofalginate microcapsules due to the presence of Ca²⁺ in the inner alginatecore, insufficient biocompatibility due to guluronic acid content inalginate/polylysine capsules, and insufficient mechanical strength ofthe alginate coating. Moreover, the process of alginate encapsulation isnonspecific and can result in the formation of microcapsules that do notcontain the cells or cell groups intended to be encapsulated or thatcontain other non-target biological materials. Due to these problems,alternative methods for cell encapsulation have been investigated.

One promising alternative to alginate crosslinking is a method termedinterfacial polymerization. Interfacial polymerization has thepossibility of offering all of the advantages of the alginateencapsulation method for cellular encapsulation and its therapeuticapplications, although there has been little done to investigate itspotential. Interfacial polymerization generally involves the formationof a layer of polymerized material, such as synthetic or naturalpolymerizable polymers, on the surface of a biological substrate. Theformation of the layer of polymeric material is generally promoted bythe activation of a polymerization initiator, which is deposited on thesurface of the biological substrate, in the presence of thepolymerizable polymers.

Some polymerization initiators for use in interfacial polymerizationmethods have been demonstrated in U.S. Pat. No. 5,410,016 and U.S. Pat.No. 5,529,914. These patents describe depositing the polymerizationinitiator, eosin Y, on a cell membrane and then activating the initiatorto promote polymerization of a macromer solution. However, the use ofeosin Y, which is a relatively nonpolar, low molecular weightlight-activated initiator dye, or compounds similar to eosin, presentsmany disadvantages for interfacial polymerization methods and alsopresents potential problems to subjects receiving transplantedencapsulated cells. For example, these dyes and other similar lowmolecular weight compounds present toxicity problems as they canpenetrate into a cell and interfere with normal biochemical pathways. Ifpenetrated into the cell, these dyes can cause free radical damage whenactivated by external sources of energy. Other drawbacks arise if thedye is able to diffuse out of the formed polymeric layer, therebyproducing potential toxicity to a host organism. Dyes such as eosin alsotend to aggregate in aqueous solution, thereby reducing the efficiencyof the encapsulation process and introducing problems withreproducibility. Finally, in view of the limited efficiency of thesedyes in initiating sufficient radical chain polymerization, it is oftennecessary to add one or more monomeric polymerization “accelerators” tothe polymerization mixture. These accelerators also tend to be smallmolecules which are capable of penetrating the cellular membrane andhave the potential to be cytotoxic or carcinogenic. Therefore, it isalso desirable to minimize the use of these accelerators. In attempts toovercome the above problems, applicants have previously introduced novelinterfacial polymerization reagents and techniques (see U.S. Pat. Nos.6,007,833 and 6,410,044; herein incorporated by reference in theirentirety).

Despite these teachings, improved initiators for interfacialpolymerization methods are desired. The cell surface, to which theinitiator polymer is targeted, is very complex and presents a challengefor the design of initiators that function in a desired manner. Forexample, the cell surface contains numerous surface proteins, some ofwhich have carbohydrate groups containing charged moieties, such assulfated proteoglycans and glycosaminoglycans. It is desirable to designinitiators that localize to the biological surface but do not affect thephysiology of the cell in a negative manner. For example, improvedinitiators should preferably promote the formation of a polymeric layeron the cell surface in an efficient manner without triggering anydetrimental cellular processes, such as signaling pathways that lead tocell death.

In another aspect, it may also be desirable to have the interfacialpolymerization reagents or polymeric layers formed by the initiatorsimpart a desired effect on the cell. For example, having encapsulatedpancreatic cells that produce insulin or having encapsulated thyroidcells produce parathyroid hormone can be of value to a patient in needof such a therapy. Such action may reduce or eliminate the need for thepatient to take drugs that promote such an effect in vivo.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for coating a surface.The method includes contacting the surface with a ligand-coupledinitiator polymer. The initiator polymer includes a polymerizationinitiator group and a ligand group, and the ligand group canspecifically bind to a receptor on the surface. The method also includescontacting the surface with a polymerizable material and then activatingthe initiator group of the ligand-coupled initiator polymer to causepolymerization of the polymerizable material on at least a portion ofthe biological surface.

The method typically involves coating a biological surface, such as theouter membrane of a cell. In one aspect, the invention provides a methodfor encapsulating pancreatic islets with a polymeric coating. Accordingto this embodiment, the ligand group used for this purpose can be asulfonylurea derivative, which can also be useful for stimulating thepancreatic β cells to secrete insulin.

In another aspect, the invention provides a ligand-coupled initiatorpolymer that includes a photoinitiator group that is selected from thegroup of light activated dyes, and a ligand group. The light activateddyes can be selected from the group consisting of acridine orange,camphorquinone, ethyl eosin, eosin Y, erythrosine, fluorescein,methylene green, methylene blue, phloxime, riboflavin, rose bengal,thionine, xanthine dyes, and the like. In another aspect, the initiatorpolymer comprises a hydrophilic backbone, such as a polyacrylamidebackbone or a backbone having similar hydrophilic properties.

The invention further provides a kit that includes an initiator polymerand a polymerizable material such as a macromer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a synthetic scheme for the preparation of asulfonylurea monomer, SUM (Compound I).

FIG. 2 illustrates a synthetic scheme for the preparation of an EITCMonomer, EITCM (Compound II).

FIG. 3 illustrates a synthetic scheme for the preparation of asulfonylurea derivative, SUNCS (Compound IV).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides ligand-coupled initiator polymers, hereinreferred to as “initiator polymers”, compositions and systems includingthe initiator polymers, and methods for performing the interfacialpolymerization of polymerizable material on a surface using theseinitiator polymers. “Polymer” refers to a compound having one or moredifferent repeating monomeric units and includes linear polymers andcopolymers, branched polymers and copolymers, such as highly brancheddendrimer polymers and copolymers, herein referred to as “dendrimers”,graft polymers and copolymers, and the like. The surface to which theinitiator polymer binds generally bears a receptor that is able toassociate with the ligand. In one embodiment of the invention thesurface is a biological surface. As used herein “biological surface”broadly refers to the surface of any sort of biological material thathas a surface receptor, such as, for example, the surface of cells, thesurface of a group of cells, the surface of biological particles such asviral particles, or the surface of tissue. The initiator polymers of theinvention are able to specifically interact with the receptor on thesurface of the biological material and promote the polymerization ofpolymerizable material to form a polymeric layer, also referred toherein as a “polymeric matrix”, on or near the biological surface.

The initiator polymers are particularly useful for cell encapsulationmethods, although they can also be used to form a matrix of polymerizedmaterial on a biological surface having a receptor in any sort of exvivo or in vivo method. Cell encapsulation involves the formation of apolymeric layer typically over the entire surface of the cell or cells,and this polymeric layer typically has certain physical and functionalproperties, such as thickness, permselectivity, strength, andprotectivity. In other embodiments, the initiator polymers can be usedto form a polymeric matrix of polymerized material on the surface of anytype of natural or synthetic material that has specific receptors thatcan interact with the ligand group of the initiator polymer.

According to the invention, initiator polymers useful for providing acoating of a polymeric material on a surface include an initiator groupand a ligand group. The initiator group refers to a portion of theinitiator polymer that can specifically accept energy and generate afree radical species, directly or indirectly, and is sufficient topromote free radical polymerization of the polymerizable material. Theligand group refers to a portion of the initiator polymer that is ableto specifically associate with a distinct receptor (for example, aligand-binding member) on the surface of the material targeted forcoating. The affinity between the ligand group and the receptor isgenerally high, typically having a dissociation constant (K_(d)) in therange of 10⁻⁶ to 10⁻¹² M.

In one embodiment, the ligand group of the initiator polymer is amolecule that can specifically associate with a therapeutically relevantreceptor on the surface of a cell. Typically, a therapeutically relevantreceptor is a receptor that can affect, either directly or indirectly,the function of a cell wherein the function is associated with changinga physical condition in a subject having the cell. For example, thebinding of the ligand group to the receptor can trigger the productionof a useful compound or can block the release of an undesirablecompound. “Production” is used in its broadest sense and includes anycellular function that causes or increases the release of thetherapeutic compound from the cell. In this particular embodiment, theligand group of the initiator polymer serves a dual function. First, theligand group specifically binds the initiator polymer to a specificreceptor on the cellular surface, and second, the ligand group provokesa cellular response from the cellular material it is encapsulating. Thecellular response can be initiated by the binding of the ligand group toits receptor, wherein the receptor moiety triggers the cell to produce adesired compound or compounds (or to elicit a desired cellularresponse). The invention also provides a novel way of initiating andmaintaining a cellular response since the ligand group of the initiatorpolymer, which becomes incorporated into the polymer matrix formed viainterfacial polymerization, remains in contact with the receptor on thecell surface following encapsulation. Therefore, the encapsulated cellcan be continuously stimulated to produce the therapeutically usefulcompound. In one aspect, the ligand group is a molecule which can bindto the surface receptor on an endocrine cell and the binding causes therelease of a compound that has an endocrine function in the body.

In a specific embodiment, the ligand group is a molecule that can bind asurface receptor on a pancreatic β cell. In some aspects, the binding ofthe molecule on the surface of the β cell can elicit a cellular responsefrom the β cell, such as the production of insulin. In a preferredembodiment the ligand group is a sulfonylurea derivative, such asglyburide. “Sulfonyl derivatives”, as used herein, refers to compoundshaving a sulfonylurea portion and that are able to produce aninsulinotropic effect. Sulfonylureas such as glyburide are ligands whichcan bind to potassium (ATP) channel proteins on the surface ofpancreatic β cells.

The ligand-coupled initiator polymer of the invention is arranged to besoluble in an aqueous solution and able to associate with the receptorbased on high affinity interactions between the ligand group and thereceptor. In some embodiments the initiator group is non-polar and insome embodiments the ligand group is also non-polar. Therefore,typically, the initiator group and the ligand group will conferhydrophobic properties to the initiator polymer. In a preferredembodiment the initiator polymer can include a polymer backbone that ishighly hydrophilic. A highly hydrophilic backbone can allow theinitiator polymer to maintain its solubility and its receptor-bindingproperties in an aqueous environment.

In one embodiment, the initiator polymer can be used in a method forcoating a biological surface, such as a cell encapsulation method. Inthese types of methods, the initiator polymer is used with apolymerizable material, such as macromers, that can form a matrix on thesurface. In some embodiments the initiator polymer is placed in contactwith the surface separately from the macromer component; in otherembodiments the initiator polymer and the macromer component are placedin contact with the surface together as a polymerizable composition tothe cells. Therefore, the invention also provides compositions thatinclude a ligand-coupled initiator polymer and a polymerizablecomponent. Other compounds useful for cell encapsulation, such asreductants/acceptors and viscosity enhancing agents can be introducedinto the polymerization method in existing steps or in additional steps.Such reagents are described in detail below. Therefore, the inventionalso provides polymerizable compositions and kits for forming a polymercoating on a surface that can include a ligand-coupled initiatorpolymer, a polymerizable component, and other components that canenhance or that are useful for coating a surface, particularly for cellencapsulation.

In a more specific embodiment the invention provides for theligand-coupled initiator polymer as a component in a group of compoundsused for interfacial polymerization methods, and applicable for thetreatment of particular diseases. These compounds and methods can beimplemented for the encapsulation of cells or tissue, wherein theencapsulated cells or tissue are therapeutically useful. Cells or tissueof a particular type can be encapsulated and introduced into a subjectin need of a certain type of cell or tissue. Endocrine cells, forexample, are one class of cells that can be encapsulated using theinitiator polymer of the invention and that can be therapeuticallyuseful following administration to a patient having an endocrine-relateddisorder. Specific types of endocrine cells such as pancreatic isletscan be encapsulated using the initiator polymer of the invention andtransplanted to a diabetic patient in need of functional pancreatictissue.

The ligand-coupled initiator polymer of the invention includes one ormore ligand groups. As used herein, “ligand group” refers to any sort ofchemical moiety that displays a specific binding interaction with areceptor on a surface. The receptor can be a molecule on a biologicalsurface (e.g., a cell surface), for example, a protein or acarbohydrate. Ligand:receptor interactions exhibit binding specificityand typically exhibit effector specificity. Specific bindinginteractions of a ligand to a receptor are generally characterized assaturable. According to the invention, ligand:receptor dissociationconstants (K_(d)) on the order of 10⁻⁶ to 10⁻¹² M are typical of mostspecific binding interactions between the ligand and receptors asdescribed herein.

The ligand group of the initiator polymer can allow for the specificlocalization and binding of the initiator polymer to the surface of abiological substrate such as a cell, group of cells, or tissue. Use ofligand groups allows for cell- or tissue-specific surface localizationof the initiator polymer and the formation of a polymeric matrix on thesurface of these specific target cells or tissues. In another aspect theligand group can serve to promote a biological response as a consequenceof the ligand:receptor interaction.

Examples of specific ligand:receptor interactions include smallmolecule:cell-surface receptor interactions such assulfonylurea:sulfonylurea receptor and amiloride:amiloride-sensitivesodium channel protein (ENaC) interactions; and protein orpeptide:cell-surface receptor interactions such as thyroid-stimulatinghormone (TSH):thyroid plasma membrane receptor, vasopressin:vasopressinreceptor, and antibody or antibody fragment:cell-surface antigeninteractions. A receptor molecule can be any sort of surface determinanton a biological material, such as a portion of a membrane protein or aportion of a carbohydrate moiety attached to membrane proteins. Theligand can be chosen to bind various classes of cell surface receptors.Such classes include, for example, G-coupled receptors, ion-channelreceptors, tyrosine kinase-linked receptors, and receptors withintrinsic enzymatic activity having one or multiple transmembranedomains.

The ligand group of the initiator polymer can be derived from any lowmolecular weight hydrophilic or lipophilic molecules; small chargedmolecules; water soluble peptides (peptide hormones); lipophilichormones including erconsanoid hormones; antibodies or antibodyfragments; proteins; and derivatives of any of the above.

The ligand group can have either an agonistic or antagonistic effect onthe biological substrate. In one embodiment, the ligand group of theinitiator polymer can bind to the receptor and elicit one or morebiological responses, such as intercellular signal transduction and geneexpression. Intercellular signal transduction can lead to, for example,changes in gene or protein expression, or changes in the modification orsecretion of a particular compound, such as a protein, from the cell. Ina preferred embodiment, the ligand is chosen to promote a biologicallyuseful response from the biological material that it is in contact with.For example, the ligand group of the initiator polymer can bind a cellsurface receptor and elicit production of a compound that isphysiologically useful, or that is therapeutic for a particular physicalcondition. The ligand group pendent from the initiator polymer can exertits biological effect alone and/or when incorporated into thepolymerized matrix that is formed after the initiator polymer isactivated.

In one particular embodiment of the invention, the ligand group of theinitiator polymer is a molecule capable of binding to a receptor on thesurface of a pancreatic β cell. In some preferred embodiments, theligand group is able to both bind the pancreatic β cell cell-surfacereceptor and stimulate an insulinotropic cellular response from thecells (for example, the production of insulin). The ligand group can bean insulinotropic agent able to cause the production and or release ofinsulin from the β cell. Therefore, according to the invention, aninitiator polymer having a pancreatic cell-binding ligand can be placedin contact with and associated with a preparation of pancreatic isletsand used to promote the formation of a matrix around the islets.Incorporated in the formed matrix is the initiator polymer containingthe ligand group. The islets encapsulated within the matrix can betransplanted to a subject, and, because of the matrix, areimmunoprotected and able to produce therapeutically useful compounds,such as insulin, that are able to produce an effect in the subject.

In one embodiment, the ligand group can associate with a portion of anATP-sensitive potassium (K⁺-ATP) channel, and can be, therefore, aK⁺-ATP channel-binding ligand. Portions of the K⁺-ATP channel caninclude, for example, K⁺-ATP channel proteins such as sulfonylureareceptor proteins SUR1, SUR2, and pore-forming subunits such as KIR6.1and KIR6.2. Particularly relevant portions of these proteins are thosethat can bind ligands which function to close the K⁺-ATP channel. Inpancreatic β cells K⁺-ATP channel-closing ligands, a subgroup of K⁺-ATPchannel-binding ligands, can function to trigger insulin secretion fromthe cells. Typically this insulin secretion is caused by the K⁺-ATPchannel-closing ligands binding and preventing potassium effluxresulting in membrane depolarization and calcium influx causing releaseof the insulin from the cells.

K⁺-ATP channel-closing ligands include first generation sulfonylureassuch as tolbutamide, tolazamide, chlorpropamide, and acetohexamide;second generation sulfonylureas such as glimepiride, glipizide, andglyburide; insulin secretagogues such as meglitinide, repaglinide,nateglinide, prandin, and starlix; imidazoline-derived drugs such asmidaglizole, LY397364, and LY389382; mitiglinide and analogues such as5-chloro-N-(2-(4-hydroxyphenyl)ethyl)-2-methoxybenzamide and4-(2-(5-chloro-2-methoxybenzamido)ethyl)phenyl phosphate (Hastedt andPanten, Biochem. Pharmacol. 65:599 (2003));9-(3,4-dichlorophenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydro-1,8(2H,5H)-acridinedione(Gopalakrishnan, M. et al. Br. J. Pharmacol. 138:393 (2003)); andfunctional derivatives thereof.

In another embodiment, the invention provides polymerizable monomershaving a ligand group that can associate with a portion of a K⁺-ATPchannel. In particular, polymerizable monomers having an ethylenicallyunsaturated group and a ligand group having a sulfonylurea portion areprovided by the invention.

The ligand groups can be coupled to the backbone of the initiatorpolymer in any suitable manner. For example, the ligand groups can becoupled to the backbone by preparing ligand-monomers and polymerizingthe ligand-monomers with initiator-monomers. Synthesis ofligand-monomers can be readily accomplished using standard chemicalreactions. Another method for preparing the initiator polymer involvespreparing a reactive ligand moiety and reacting the ligand moiety with areactive group on a preformed polymer. For example, an isocyanate orisothiocyanate derivative of a ligand group can be reacted with apolymer containing pendent amine groups thereby forming an initiatorpolymer bearing pendent ligand groups. The ligand groups can be coupledto and spaced in any suitable manner along the length of the polymerbackbone, for example the ligand groups can be spaced in a random orordered pattern along the length of the polymer backbone chosen or canbe present primarily on one end of the polymer backbone.

The number of ligand groups coupled to the polymer backbone can bearranged to provide an initiator polymer that associates with the cellsurface in a manner suitable to allow the formation of a polymericmaterial on the surface when the initiator polymer is activated. In oneembodiment, the initiator polymer includes at least one ligand group. Inanother embodiment of the invention, the initiator polymer has up toabout 5% of the monomeric units of the polymer coupled to ligand groups.In yet another embodiment the initiator polymer has up to about 10% ofthe monomeric units of the polymer coupled to ligand groups.

According to the invention, the ligand-coupled initiator polymerincludes one or more initiator groups, which are coupled to the backboneof the initiator polymer. The initiator groups are able to promote freeradical polymerization of polymerizable material, such as macromers,when energy capable of activating the initiator group is applied to theinitiator polymer. Activated initiator groups can cause free radicalpolymerization of the polymerizable material either directly orindirectly. Indirect methods typically include the transfer of energyfrom the activated initiator to an acceptor or reductant, a chemicalspecies that can form a free radical and can act to cause polymerizationof the polymerizable material. In direct methods the initiator groupprovides the free radical itself.

According to the invention, the initiator polymer can be localized to asurface, such as the surface of a cell, via interaction of the ligandgroups with the receptor on the surface. Upon activation of theinitiator groups, polymerizable material that is in proximity to theinitiator polymer polymerizes, leading to the formation of a layer ofpolymeric material, or a matrix, on the surface. This type ofpolymerization is typically referred to as interfacial polymerization.

The initiator polymer can include light-activated photoinitiator groups,thermally activated initiator groups, chemically activated initiatorgroups, or combinations thereof. Suitable thermally activated initiatorgroups include 4,4′azobis(4-cyanopentanoic) acid and2,2-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride or otherthermally activated initiators provided these initiators can beincorporated into an initiator polymer. Chemically activated initiationis often referred to as redox initiation, redox catalysis, or redoxactivation. In general, combinations of organic and inorganic oxidizers,and organic and inorganic reducing agents are used to generate radicalsfor polymerization. A description of redox initiation can be found inPrinciples of Polymermization, 2^(nd) Edition, Odian G., John Wiley andSons, pgs 201-204 (1981). Redox initiators that are not damaging tobiological systems are preferably used. Photoinitiator groups andthermally activated initiator groups that utilize energy that is notdamaging to biological systems are preferably used. In one embodiment,photoinitiator groups having long wavelength UV and visiblelight-activated frequencies are coupled to the backbone of the initiatorpolymer. In a preferred embodiment, visible light-activatedphotoinitiators are coupled to the polymer backbone.

Photoinitiation can occur by various mechanisms, including Norrish typeI reactions, intra- or intermolecular hydrogen abstraction reactions,and photosensitization reactions utilizing photoreducible orphoto-oxidizable dyes. The latter two types of reactions are commonlyused with an energy transfer acceptor or a reductant, which can be, forexample, a tertiary amine. Such tertiary amines can be incorporated intothe polymeric backbone of the macromer. In a preferred embodiment, theinitiator polymer includes one or more initiator groups that allow forintra- or intermolecular hydrogen abstraction reactions orphotosensitization reactions utilizing photoreducible orphoto-oxidizable dyes when activated. Useful energy transfer acceptorsor reductants for use with these types of initiators include, but arenot limited to, tertiary amines such as triethanolamine, triethylamine,N-methyl diethanolamine, N,N-dimethyl benzylamine, tetramethylethylenediamine; secondary amines such as dibenzyl amine, N-benzylethanolamine, N-isopropyl benzylamine; and primary amines such asethanolamine, lysine, and ornithine.

In one embodiment, photoinitiator groups having an absorbance of 350 nmand greater are used. More preferably, photoinitiator groups having anabsorbance of 500 nm and greater are used. Suitable photoinitiatorgroups include light-activated initiator groups, such as long-wave ultraviolet (LWUV) light-activatable molecules and visible light activatablemolecules. Suitable long-wave ultra violet (LWUV) light-activatablemolecules include, but are not limited to,[(9-oxo-2-thioxanthanyl)-oxy]acetic acid, 2-hydroxythioxanthone, andvinyloxymethylbenzoin methyl ether. Suitable visible light activatablemolecules include, but are not limited to acridine orange,camphorquinone, ethyl eosin, eosin Y, erythrosine, fluorescein,methylene green, methylene blue, phloxime, riboflavin, rose bengal,thionine, xanthine dyes, and the like.

One common feature of these visible light activatable photoinitiatorgroups, and photoinitiator groups in general, is that of having anonpolar portion. Due to the presence of this nonpolar portion, thesephotoinitiator groups generally have a low solubility in aqueoussolutions. When these photoinitiator groups are coupled to anothermolecule, such as a polymer, the photoinitiator groups can confernonpolar characteristics to the polymer conjugate and can generallyreduce the solubility of the polymer conjugate in an aqueous solution.

The initiator polymer is coupled to a number of initiator groups in anamount sufficient to promote free radical polymerization ofpolymerizable material on a surface, such as the surface of a cell. Theinitiator polymer contains at least one and more typically a pluralityof initiator groups. In some cases, the initiator polymer is highlyloaded with initiator groups and can provide a high level ofpolymerization initiator activity. This may be desirable in caseswherein the number of receptor molecules on the surface of a cell is lowand the highest polymerization potential per initiator polymer isdesired. In another aspect, an initiator polymer highly loaded withinitiator groups can be prepared and used in methods or compositionsthat include macromers that do not readily polymerize to form apolymeric layer. Accordingly, the invention provides ligand-coupledinitiator polymers that are highly loaded with initiator groups.

According to the invention, the initiator polymer includes at least oneinitiator group. In another embodiment of the invention, the initiatorpolymer has up to about 5% of the monomeric units of the initiatorpolymer coupled to initiator groups. In yet another embodiment, about10% of the monomeric units of the polymer are coupled to initiatorgroups. The initiator groups can be coupled to and pendent along thepolymer backbone at any position and can be spaced in a random orordered manner. The initiator groups preferably do not interfere withthe ability of the initiator polymer to specifically associate with itsreceptor on a surface, such as a cell surface.

The initiator group can be coupled to the initiator polymer using anysuitable method. In one method, for example, polymerizable monomershaving initiator groups can be synthesized and subsequently used in apolymerization reaction to create an initiator polymer with pendentinitiator groups. Synthesis of initiator-derivatized monomers can bereadily accomplished using standard chemical reactions. For example, anisothiocyanate or an acid chloride analog of a photoinitiator group,such as a light-activated dye, can be reacted with an ethylenicallyunsaturated amine-containing monomer to form an initiator-derivatizedmonomer. In another method of preparing the initiator polymer, preformedpolymers having reactive groups are reacted with initiator groups toattach the initiator groups to the preformed polymer. For example, anisothiocyanate analog of a photoinitiator can be reacted with a polymerhaving pendent amine groups thereby forming an initiator polymer havingpendent initiator groups. Other synthetic schemes known to those skilledin the art can be employed to prepare the initiator polymer. Theseschemes are contemplated but will not be discussed in further detail.

In preferred embodiments the initiator polymer includes a plurality ofinitiator groups that are typically nonpolar. The presence of aplurality of initiator groups can confer substantial hydrophobicproperties to the initiator polymer. Accordingly, this substantialhydrophobic property can be counter balanced by providing the initiatorpolymer with a hydrophilic backbone, which is discussed in detail below.

In a preferred embodiment of the invention, the ligand-coupled initiatorpolymer includes a ligand group, an initiator group, and is soluble inan aqueous solution. Generally, the initiator polymer includes ahydrophilic polymer backbone. The polymer backbone, which generallyrefers to the polymer chain without addition of any initiator group orligand group, typically includes carbon and preferably one or more atomsselected from nitrogen, oxygen, and sulfur. The backbone can includecarbon-carbon linkages and, in some preferred embodiments, can alsoinclude one or more of amide, amine, ester, ether, ketone, peptide, orsulfide linkages, or combinations thereof.

The polymeric backbone of the initiator polymer can include chemicalgroups useful for coupling the ligand group and the initiator group tothe backbone to form the initiator polymer. Suitable chemical groupsinclude acid (or acyl) halide groups, alcohol groups, aldehyde groups,alkyl and aryl halide groups, amine groups, amide groups, carboxylgroups, and the like. These chemical groups can be present either on apreformed polymer or on monomers used to create the ligand-coupledinitiator polymer. Examples of polymers having suitable reactive orcharged side group include polymers having reactive amine groups such aspolylysine, polyornithine, polyethylenimine, and polyamidoaminedendrimers.

In one embodiment of the invention, the backbone of the initiatorpolymer provides the initiator polymer with hydrophilic properties.Preferred hydrophilic backbones include highly water-soluble polymerssuch as polyacrylamide. Examples of suitable polymer backbones includepolyesters, polycarbonates, polyamides, polyethers (such aspolyoxyethylene), polysulfones, polyurethanes, and copolymers containingrepresentative monomer groups. Other suitable polymers includepolyamines such as polyethylenimine, polypropylenimine, and the like,and polyamine polymers or copolymers formed from monomers such as2-aminoethylacrylate, N-(3-aminopropyl)methacrylamide, and diallylamine. In one preferred embodiment the backbone of the initiator polymercontains relatively few or no aromatic groups. Therefore, in onepreferred embodiment of the invention, the initiator polymer includes(i) a polymerization initiator group, (ii) a ligand group, and (iii) ahydrophilic backbone.

In another aspect, the hydrophilic character of the initiator polymercan be improved by coupling charged groups to the polymer backbone. Inthese embodiments it is preferable that the initiator polymer isconfigured so that the presence of the charged groups does not interferewith the ability of the initiator polymer to associate with the targetreceptor on the surface of the substrate to be coated. Suitable chargedgroups include cationic groups such as quaternary ammonium, quaternaryphosphonium, and ternary sulfonium groups. Suitable anionic groups thatcan be coupled to the initiator polymer include, but are not limited to,sulfonate, phosphonate, and carboxylate groups.

An initiator polymer having at least one initiator group and at leastone ligand group can be prepared a variety of ways. For example, theinitiator group and the ligand group can be attached to a “preformed”polymer or a copolymer that is reactive with the initiator and ligandgroups. The preformed polymer or copolymer can be obtained from acommercial source or be synthesized from the polymerization of a desiredmonomer or combination of different monomers. In one example ofpreparing the initiator polymer, the initiator groups and the ligandgroups are reacted with and attached to, for example, by covalentbonding, chemical groups pendent from the backbone of the polymer orcopolymer. Such attachments of the initiator groups and the ligandgroups can be achieved by, for example, substitution or additionreactions.

In another method of preparing the initiator polymer, monomers havinginitiator and monomers having ligand groups are first prepared. Theseinitiator and ligand group-containing monomers are then co-polymerizedto create an initiator polymer having both initiator and ligand groups.In some embodiments an individual monomer having both an initiator groupand a ligand group can be used to prepare the initiator polymer.Optionally, other monomers that are not coupled to either an initiatoror cationic groups can be polymerized with the ligand andinitiator-coupled monomers to create the initiator polymer. A usefulmixture of monomers for preparation of the initiator polymer includes upto about 10 wt % of a ligand-monomer, up to about 90 wt % of ahydrophilic monomer, and up to about 20 wt % of a monomer having acharged group. Methods of preparing the initiator polymer areexemplified below. Other standard methods known to those of skill in theart to prepare the initiator polymer are contemplated and will not bediscussed further.

In one embodiment, the initiator polymer has (i) an amount of ligandgroups that allow the initiator polymer to specifically associate with areceptor on a surface, (ii) an amount of initiator groups that canpromote polymerization of a macromer on a surface, and (iii) ahydrophilic backbone of a size sufficient to solubilize the initiatorpolymer in an aqueous solution. In various embodiments, the initiatorpolymer has a weight average molecular weight (M_(w)) of greater thanabout 50K Da, 100K Da, 250K Da, 500K Da, 750K Da, and 10,000 K Da. Insome embodiments it is preferable that the initiator polymer has aM_(w), in the higher ranges of these molecular weights recited.

As used herein “weight average molecular weight” or M_(w), is anabsolute method of measuring molecular weight and is particularly usefulfor measuring the molecular weight of a polymer (preparation), such aspreparations of initiator polymers and macromers. Polymer preparationstypically include polymers that individually have minor variations inmolecular weight. Polymers are molecules that have a relatively highmolecular weight and such minor variations within the polymerpreparation do not affect the overall properties of the polymerpreparation (for example, the characteristics of an initiator polymerpreparation). The weight average molecular weight (M_(w)) can be definedby the following formula:

$M_{w} = \frac{\sum\limits_{i}\; {N_{i}M_{i}^{2}}}{\sum\limits_{i}\; {N_{i}M_{i}}}$

wherein N represents the number of moles of a polymer in the sample witha mass of M, and Σ_(i) is the sum of all N_(i)M_(i) (species) in apreparation. The M_(w) can be measured using common techniques, such aslight scattering or ultracentrifugation. Discussion of M_(w) and otherterms used to define the molecular weight of polymer preparations can befound in, for example, Allcock, H. R. and Lampe, F. W., ContemporaryPolymer Chemistry; pg 271 (1990).

Therefore, in one specific embodiment of the invention, the initiatorpolymer includes (i) a plurality of polymerization initiator groups,(ii) a ligand group, and (iii) a hydrophilic backbone, wherein the M_(w)of the initiator polymer is greater than about 50K Da, more preferablygreater than about 100K Da, and most preferably greater than about 250KDa.

In another specific embodiment of the invention, the initiator polymerincludes (i) a plurality of photoinitiator groups selected from thegroup of visible light-activated dyes, (ii) a ligand group, and (iii) ahydrophilic backbone, wherein the M_(w) of the initiator polymer isgreater than about 50K Da, more preferably greater than about 100K Da,and most preferably greater than about 250K Da.

In yet another specific embodiment of the invention, the initiatorpolymer includes (i) a plurality of photoinitiator groups selected fromthe group of visible light-activated dyes, (ii) a K⁺-ATP channel-bindingligand group, and (iii) a hydrophilic backbone, wherein the M_(w) of theinitiator polymer is greater than about 50K Da, more preferably greaterthan about 100K Da, and most preferably greater than about 250K Da.

The ligand-coupled initiator polymer can promote the polymerization ofpolymerizable material, such as macromers, on a surface having aligand-binding receptor. A matrix of polymeric material is formed on thesurface after the initiator polymer is activated. The polymerizablematerial can be any sort of compound, including monomers and polymershaving one or more polymerizable groups. Polymerizable groups areportions of the polymerizable compounds that are able to propagate freeradical polymerization, such as carbon-carbon double bonds. Preferredpolymerizable groups are found in polymerizable compounds having vinylor acrylate groups. More specific polymerizable portions includeacrylate groups, methacrylate groups, ethacrylate groups, 2-phenylacrylate groups, acrylamide groups, methacrylamide groups, itaconategroups, and styrene groups. Preferred materials for the encapsulation ofcellular material are biocompatible polymerizable polymers (alsoreferred to as macromers). Such macromers can be straight chain orbranched polymers or copolymers, or graft copolymers. Syntheticpolymeric macromers, polysaccharide macromers, and protein macromerssuitable for use with the initiator polymer of the current invention aredescribed in U.S. Pat. No. 5,573,934 (Hubbell et al.), the teaching ofwhich is incorporated in its entirety by reference.

Preferred macromers include, but are not limited to, polymerizablepoly(vinylpyrrolidone) (PVP), poly(ethylene glycol) (PEG), poly(ethyleneoxide) poly(ethyloxazoline), poly(propylene oxide), polyacrylamide(PAA), poly(vinyl alcohol) (PVA), copolymers thereof, and the like. Inparticular, PEG and PAA are more preferred macromers. These types ofmacromers are typically soluble in water and are more stable in vivo ascompared to biodegradable polymers.

In some cases it may be desirable to use naturally occurring orsynthetic macromers as the polymerizable material. Suitable macromersinclude naturally occurring polymers such as polysaccharides, examplesof which include, but are not limited to, hyaluronic acid (HA), starch,dextran, heparin, and chitosan; and proteins (and other polyaminoacids), examples of which include, but are not limited to, gelatin,collagen, fibronectin, laminin, albumin, and active peptides thereof. Inorder to make these naturally occurring or synthetic macromerspolymerizable, polymerizable groups can be incorporated into a polymerusing standard thermochemical reactions. For example, polymerizablegroups can be added to collagen via reaction of amine containing lysineresidues with acryloyl chloride. These reactions result in collagencontaining polymerizable moieties. Similarly, when synthesizing amacromer, monomers containing reactive groups can be incorporated intothe synthetic scheme. For example, hydroxyethylmethacrylate (HEMA) oraminopropylmethacrylamide (APMA) can be copolymerized withN-vinylpyrrolidone or acrylamide yielding a water-soluble polymer withpendent hydroxyl or amine groups. These pendent groups can subsequentlybe reacted with acryloyl chloride or glycidyl acrylate to formwater-soluble polymers with pendent polymerizable groups. Suitablesynthetic polymers include hydrophilic monomers containing degradablesegments as described in U.S. Pat. No. 5,410,016 supra, the teaching ofwhich is incorporated in its entirety by reference.

In another aspect, the invention provides a polymerizable compositionthat includes a ligand-coupled initiator polymer and a macromer. Thepolymerizable composition can also include other compounds useful cellencapsulation methods such as reductant/acceptors andviscosity-enhancing agents, for example, polyethylene glycols, andglycerol. Therefore, in one embodiment, the invention provides apolymerizable composition that includes: (i) an initiator polymer havingat least one polymerization initiator group and a ligand group that iscapable of interacting with a receptor on a surface, and (ii) amacromer. In a more specific embodiment the invention provides apolymerizable composition that includes: (i) an initiator polymer havingphotoinitiator group selected from the group of visible light-activateddyes, and a ligand group able to interact with a receptor on a surface,and (ii) a macromer.

Cell Encapsulation Methods

As previously indicated, the initiator polymer of the invention istypically used with macromers and, in some cases, a reductant/acceptorin a method to provide a coating to a biological surface. The reagentsare particularly suitable for cell encapsulation processes.

Cells or tissue to be encapsulated can be obtained from an organism, forexample, a human donor, or obtained from a cell culture, which can betransformed or otherwise modified. Specific types of cells and tissuethat can be encapsulated and used for the treatment of diseases arediscussed below. “Cells” refers to individual membrane-bound biologicalunits that can be present as part of a tissue or organ, or can functionindependently as micro-organisms. “Tissue” refers to a biological massthat includes groups of similar cells, and also typically includesextracellular material that is associated with the cells. Cells, ortissue in particular, can be subject to treatment prior to theencapsulation process. For example, tissue can be treated with enzymaticor other suitable reagents, such as trypsin, hyaluronidase, orcollagenase, to obtain individual cells or cell groups of a suitablesize for the encapsulation process. Alternatively, tissue can be subjectto mechanical processes in order to prepare suitable cellular startingmaterial. Prior to encapsulation cells can also be treated with drugs,prodrugs, hormones, or the like, or can be cultured to provide cellsthat display a desired expression pattern or have a certainmorphological features. Technical references that provide detailedinstructions for the preparation of cells or tissue and the treatment ofprepared cells or tissue are available and can be found in, for example,in Basic Cell Culture Protocols, Pollard, J. W. and Walker, J. M., Ed.(1997).

Alternatively, cells or tissue suitable for encapsulation and intendedfor use with the ligand-coupled initiator of the invention can becommercially obtained. For example, viable human liver preparations suchas microsomes and hepatocytes, and viable human pancreatic preparationssuch as pancreatic islets, can be obtained from commercial sources suchas CellzDirect, Inc. (Tucson, Ariz.).

With information available in technical literature, one can utilize theligand-coupled initiator polymer in methods for coating a surface, andin particular, in the novel and inventive methods as described hereinfor encapsulating cells and tissue. For example, the teaching Cruise, etal., Cell Transplantation 8:293 (1999), can provide a basis for the cellencapsulation methods using the ligand-coupled initiator polymer of theinvention. Cells or tissue suitable for the encapsulation process,prepared as indicated above or obtained from a commercial source, can besuspended in a suitable solution, such as a biocompatible bufferedaqueous solution, such as, for example Roswell Park Memorial Institute(RPMI) media. Other reagents can be added to this solution, such asanimal serum; proteins such as albumin; oxidants; reductants; vitamins;minerals; growth factors; or other components that can have an impact onthe viability and function of the cells or tissues.

The ligand-coupled initiator polymer can be added to this solutionbefore or after contacting the cells or tissue with the solution. Theinitiator polymer can be brought into contact with the cells in anamount that is sufficient for formation of a matrix around the cells ortissue. In one embodiment, the concentration of the initiator polymer isfrom 0.001 to 0.5 wt %. In yet another embodiment, the concentration ofthe initiator polymer is from 0.1 to 0.25 wt %. In one embodiment theinitiator polymer is brought in contact with the cells for a period oftime that is sufficient for the initiator polymer to associate with thesurface of the cells. Optionally, a washing step can be performed. Thiswashing step can be used, for example, to remove excess unboundinitiator or other material in contact with the cells. After theinitiator polymer is brought in contact with the cells or tissue, thepolymerizable material, such as macromers, can be brought in contactwith the cells. In another embodiment, the initiator polymer is broughtinto contact with the cells or tissue together with the polymerizablematerial. In yet another embodiment the polymerizable material isbrought into contact with the cells prior to bringing the initiatorpolymer into contact with the cells.

The polymerizable material (e.g., macromers) can be brought into contactwith the cell or tissue in an amount that allows formation of a matrixof a desired thickness. A concentration of macromer in solution usefulfor cell encapsulation can be in the range of 5-50 wt %, and morepreferably in the range of 10-30 wt %. In some embodiments, thepolymerizable material can be placed in contact with the cells for aperiod of time prior to activating the ligand-coupled initiator polymer.

Other reagents can be brought in contact with the cells or tissue duringthe encapsulation process. As previously mentioned, such reagentsinclude acceptors or reductants, such as tertiary amines (e.g.,triethanolamine) that can form a free radical and cause free radicalpolymerization of the polymerizable material. Suitable acceptors orreductants are known in the art and are commercially available. Theseacceptors or reductants are typically used in indirect polymerizationmethods wherein the initiator group transfers energy to the acceptors orreductants to promote free radical polymerization of the polymerizablematerial. Reagents such as viscosity-enhancing reagents can also be usedin the method of the invention. Viscosity-enhancing reagents can improvethe process of polymerization. Suitable viscosity-enhancing reagents areknown in the art and are commercially available. One of skill in the artcan determine suitable amounts of any of these additional reagents forperforming the encapsulation process.

After the reagents necessary to promote formation of a matrix arebrought in contact with the surface to be coated, a source of energy,such as a thermal or electromagnetic energy sufficient to activate theinitiator group, is applied to initiate polymerization of thepolymerizable material. Long-wave ultra violet (LWUV) and visiblewavelengths in range of 350 nm to 900 nm are preferred and can besupplied by lamps and laser light sources. Lamps or laser light sourcesthat can provide these wavelengths of light are commercially availableand can be obtained from, for example, EFOS Inc. (Mississauga, Ontario,Canada). A particularly suitable wavelength for use with the preferredinitiator polymers of the invention is about 520 nm. The time andtemperature of the reaction are maintained to provide a desired coating.For example, the cells or tissue in contact with the initiator polymerand macromer can be treated with light for a period in the range ofseconds to minutes. The polymerization reaction can be terminated byremoving the light source. The encapsulated cells or tissue can then besubject to further treatment if desired. For example, it may bedesirable to concentrate the encapsulated material, for example, bycentrifugation, prior to introducing the encapsulated material into asubject.

As indicated, a number of technical references that provide detailedprocedures for encapsulating cells are available and can provide aframework for which the ligand-coupled initiator polymer can be used.Therefore using the available information, one can perform surfacecoating of a material, more specifically, the encapsulation of cellularmaterial and tissue using the ligand-coupled initiator polymer andreagents described herein or in other references.

Treatment

According to the invention, the initiator polymer can be used to promotethe formation of a matrix of polymerized material on a biologicalsurface. Polymerization using the initiator polymer can be performed invivo by applying an initiator polymer and polymerizable material, eithertogether or separately, to a subject in either an invasive or in anoninvasive procedure. Other particularly useful applications involvethe ex vivo encapsulation of cells or tissue. In this application cellsor tissue can be obtained from a suitable source, encapsulated with amatrix of polymeric material using a composition including the initiatorpolymer described herein, and then introduced into a subject in need ofthe encapsulated cells or tissue. In some cases, after receiving thetransplanted encapsulated cells, the subject can be administered apharmaceutical agent, such as a compound that is different than thecompound used as the ligand group of the initiator polymer, that canpenetrate the matrix that encapsulates the cells and can provoke acellular response which is of therapeutic value to the subject. Thistype of ex vivo encapsulation and transplantation procedure isadvantageous as it can provide a matrix coating affording thetransplanted cells protection from host immune rejection while allowingthe encapsulated cells to provide a therapeutic value to the host.

In one aspect of the invention, the initiator polymer is used toencapsulate cells or tissue from glands and organs of the endocrinesystem, which include cells from the pituitary gland; cells from theadrenal gland; cells from the thyroid/parathyroid glands; cells from thepancreatic islets, such as beta cells, alpha cells, delta cells, andpancreatic polypeptide (PP) cells; cells from the liver; and cells fromreproductive glands such as the testis and ovary. Endocrine cells can beremoved from a donor individual and encapsulated with polymeric materialusing the initiator polymer as described herein.

Encapsulated endocrine cells can be transplanted to an individual havingany of the following conditions or needs: a pituitary disorder and inneed of growth hormone (GH), adrenocorticotropic hormone (ACTH),follicle stimulating hormone (FSH), leutinizing hormone (LH), thyroidstimulating hormone (TSH), oxytocin, or antidiuretic hormone (ADH); anadrenal disorder and in need of mineralcorticoids (for example,aldosterone) glucocorticoids (for example, cortisol), androgenicsteroids, or catecholamines such as epinephrine or norepinephrine; athyroid or parathyroid disorder and in need of thyroxin, calcitonin, orparathyroid hormone (PTH); a pancreatic disorder such as diabetes and inneed of insulin, glucagon, somatostatin, or pancreatic polypeptide; aliver disorder and in need of bile or plasma proteins, includingclotting factors; a reproductive gland disorder and in need of malehormones such as testosterone or female hormones such as estrogen.

Other types of cells that can be encapsulated include immature andmature cells from the cardiovascular, respiratory, renal, nervous,muscular, and skeletal systems. In some aspects cells that have beentransformed or genetically modified can be encapsulated and transplantedinto a host. For example, cells that have been transformed or modifiedto produce a therapeutically useful compound, such as a peptide hormoneor an enzyme can be encapsulated and introduced into an individual.

The invention also specifically provides interfacial polymerizationcompounds, compositions, and methods for the treatment of diabetes. Inparticular, the invention provides for initiator polymers useful for thebinding to and promoting the interfacial polymerization of abiocompatible polymeric layer around pancreatic β cells and islets. Atthe same time, the use of the initiator polymer to provide the polymericlayer stimulates a desirable cellular response by potentiating anincreased insulin production from the cells.

As stated above, in some instances, a pharmaceutical agent can beadministered to the subject after transplantation of the encapsulatedcells. The pharmaceutical agent can provoke a therapeutically usefulcellular response from the encapsulated cells if needed. Other drugsthat can stimulate insulin production and that can be coadministeredwith the transplanted encapsulated β cells include metformin, acarbose,and troglitazone. Other useful drugs include that can be administered tosubjects having encapsulated cells include antithrombogenic,anti-inflammatory, antimicrobial, antiproliferative, and anticancercompounds, as well as growth factors, morphogenic proteins, and thelike.

In another aspect, the initiator polymer and polymerizable material canalso be used in in vivo applications to provide artificial barriers, forexample, barriers to prevent tissue adhesion following surgery. For thisapplication, the initiator polymer along with polymerizable material isapplied to the surface of the tissue. The composition is thenilluminated to initiate polymerization and a bather matrix is formed.The polymeric matrix prevents other tissue from adhering to the coatedtissue. In some procedures a polymeric matrix can be formed on thesurface of a blood vessel to prevent blood factors or cells, such asplatelets, from interacting with or adhering to the blood vessel wall.Both degradable and non-degradable macromer systems can be used for thispurpose.

The initiator polymer of the invention can also be utilized for othermedically useful purposes. For example, the initiator polymer can be acomponent used for forming adhesives for tissue and other surfaces. Inanother example, the initiator polymer can be applied to a surfacebearing a receptor, to which adhesion is desired. The surface can bewashed to remove any unbound or excess initiator polymer and apolymerizable material can be added. Another surface can be contactedwith the initiator polymer-coated surface and then a source of energycan be applied to activate the initiator polymer and to polymerize thepolymerizable material, thereby forming a surface-to-surface junction.If a temporary adhesive is desired, the polymerizable material caninclude a degradable material, for example, biodegradable macromers.

The initiator polymer can also be used for the formation of barriers onsurfaces bearing a receptor. An example of such an application is abarrier for the prevention of tissue adhesion following surgery. Forthis application, an initiator polymer can be applied to the surface ofdamaged tissue. The surface can be washed to remove unbound or excessinitiator polymer, and a polymerizable material can then be added. Theinitiator polymer can then be activated on the surface to polymerize thepolymerizable material. The polymeric matrix formed by thispolymerization can prevent other tissue from adhering to the damagedtissue. Both degradable and/or non-degradable macromers can be used inthis barrier formation method.

The invention will now be demonstrated referring to the followingnon-limiting examples.

TABLE I

(Compound I: SUM)

(Compound II: EITCM)

(Compound III: SEAA Initiator Polymer)

(Compound IV: SUNCS)

(Compound V: SEAP Initiator Polymer)

EXAMPLES Example 1 Synthesis of a Sulfonylurea Monomer (SUM)

Preparation of a monomer having a sulfonylurea ligand portion isachieved according to the synthetic scheme as illustrated in FIG. 1. Asolution of 4-(2-aminoethyl)benzenesulfonamide (AEBS) and triethylamine(TEA) in chloroform (or acetonitrile) is cooled in an ice bath. Asillustrated in step 1 of FIG. 1, to the cooled stirred AEBS solution isadded a solution of methacryloyl chloride (MAC), in chloroform (oracetonitrile). After the addition is completed, the reaction is stirredat room temperature for 2 hours. The volatile organic materials areremoved under vacuum with an air bleed to avoid polymerization. Theresidue (sulfamoyl monomer intermediate (SMI):2-methyl-N-(2-(4-sulfamoyl-phenyl)-ethyl)-acrylamide) is dissolved in anaqueous sodium hydroxide solution. As illustrated in step 2, to theaqueous solution is added a solution of cyclohexyl isocyanate (CI) inacetone (or acetonitrile), and the resultant reaction is stirred at roomtemperature for 16 hours. Finally, the reaction is acidified with HCland the precipitate is isolated and dried to give the sulfonylureamonomer (Compound I: SUM), which is also shown in Table I.

Example 2 Synthesis of an EITC Monomer (EITCM)

Preparation of a monomer having an EITC photoinitiator portion isachieved according to the synthetic scheme as illustrated in FIG. 2. Toa solution of eosin isothiocyanate (EITC;4-Isothiocyanato-2-(2,4,5,7-tetrabromo-6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoicacid methyl ester) in dimethylsulfoxide (DMSO) is added a solution ofN-(3-aminopropyl)methacrylamide (APMA) in chloroform. The solution isstirred at room temperature for 16 hours. The chloroform is removedunder vacuum with an air bleed. EITCM in a DMSO solution is used in thepreparation of an initiator polymer in Example 3. EITCM (Compound II) isalso shown in Table I.

Example 3 Preparation of a SUM-EITCM-AMPS-Acrylamide (SEAA) InitiatorPolymer

The initiator polymer, which can be represented by Compound III asillustrated in Table I, is prepared by placing SUM, EITCM, AMPS (sodium2-acrylamido 2-methyl propane sulfonate), mercaptoethanol, AIBN(2,2′-azobisisobutyronitrile) and DMSO (dimethylsulfoxide) in a glassvessel and polymerizing the mixture. The solution is degassed(deoxygenated), blanketed with argon and heated at 55° C. with stirringfor 16 hours. The DMSO solution containing the polymer product is placedin 12-14 kDa molecular weight cut off (MWCO) dialysis tubing anddialyzed against deionized water. The product is then isolated bylyophilization.

Example 4 Synthesis of a Sulfonylurea-Isothiocyanate (SUNCS)

A sulfonylurea derivative (SUNCS) is synthesized in preparation formaking another sulfonylurea-containing initiator polymer. Thepreparation of SUNCS is achieved according to the synthetic scheme asillustrated in FIG. 3. 4-(2-Amino-ethyl)-benzenesulfonamide (AEBS) isfirst dissolved in acetonitrile (or chloroform). In step 1 the AEBSsolution is reacted with carbon disulfide (CS₂) anddicyclohexylcarbodiimide (DCC). Dicyclohexylurea (DCU) product isremoved from sulfamoyl-isothiocyanate (S-NCS) by filtration. Thesolvents are then removed to give the S-NCS intermediate. In step 2 theS-NCS, cyclohexyl isocyanate, and tetrabutylammonium bromide (TBAB) areplaced in a glass vessel with tetrahydrofuran (THF) and the mixture isstirred under an inert dry atmosphere during the slow addition of sodiumhydride (NaH). The SUNCS product (Compound IV, also shown in Table I) isthen isolated and purified by flash chromatography.

Example 5 Preparation of a SUNCS-EITC-APTAC-PEI (SEAP) Initiator Polymer

A SEAP initiator polymer is prepared according to the followingprocedure. DMSO solutions containing eosin isothiocyanate (EITCNCS;Sigma-Aldrich Corp., St. Louis, Mo.), acrylamido propyltrimethylammonium chloride (APTAC; Sigma-Aldrich Corp., St. Louis, Mo.) and SUNCSare first individually prepared. A solution containing polyaminepolyethylenimine (PEI) having a M_(w) of 10,000 Da is prepared bydissolving the PEI in DMSO. To the PEI solution is added the EITCNCS,APTAC, and SUNCS solutions. The reaction is stirred for 16 hours at roomtemperature. The polymerization product is purified using 5,000 MWCOdialysis tubing and the product is isolated by lyophilization. The finalproduct can be represented by Compound V in Table I wherein EITCrepresents eosin, APTAC represents acrylamido propyltrimethyl ammoniumchloride, and SU represents sulfonylurea.

1. A ligand-coupled initiator polymer comprising: a) a hydrophilicpolymeric backbone; b) a photoinitiator group selected from the groupconsisting of acridine orange, camphorquinone, ethyl eosin, eosin Y,erythrosine, fluorescein, methylene green, methylene blue, phloxime,riboflavin, rose bengal, thionine, and xanthine dyes; and c) a ligandgroup that can specifically bind to a receptor.
 2. A kit comprising: a)a ligand-coupled initiator polymer comprising: i) an initiator group;and ii) a ligand group able to specifically bind to a receptor on asurface; and b) polymerizable material able to form a polymeric layer onthe surface upon activation of the initiator group.